PURPOSE: Despite growing evidence of the link between elevated levels of trimethylamine-N-oxide (TMAO) and multiple diseases, there is no method with which to spatially monitor its hepatic formation from the interstitially produced trimethylamine (TMA). This study aimed to develop a deuterium metabolic spectroscopy (DMS) and imaging (DMI) approach to detect the TMA-to-TMAO metabolism in vivo. METHODS: The metabolism of (2)H(9)-TMA (TMA-d(9)) to (2)H(9)-TMAO (TMAO-d(9)) in cells overexpressing the hepatic enzyme flavin-dependent monooxygenase 3 (FMO3) was monitored in vitro with (2)H-NMR. Using an ultrahigh-field (15.2T) MRI scanner, the hepatic metabolism of the orally administered TMA-d(9) to TMAO-d(9) was studied in mice with DMS and DMI. RESULTS: The spectrally resolved (2)H-NMR peaks of intracellularly produced TMAO-d(9) (3.1 ppm) from that of supplemental TMA-d(9) (2.7 ppm) could be detected only in cells that overexpressed FMO3. In vivo, DMS and DMI experiments performed after oral administration of TMA-d(9) revealed the conversion to high TMAO-d(9) levels in the liver of females, which express high levels of FMO3. In contrast, there was no indication of TMAO-d(9) production in the liver of males, in agreement with reports of the role of testosterone in downregulating the expression of FMO3. CONCLUSION: This work shows the ability to use (2)H-MR-based methodologies to spatially monitor the TMA-to-TMAO metabolic pathway in vivo, and thus should be explored further to investigate the role of TMAO in diverse pathologies.